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15/12/2016
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1
FOSTERING GLOBAL RESPONSIBLE RESEARCH
WITH CRISPR-Cas9 Hervé Chneiweiss
Buenos Aires 1st october 2016
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• An increadible pace of scientific discoveries and technical breaks:
• CRISPR-Cas9 but also C2c2 and more
• The human genome for less than $1000 (exome for less than 400$)
• In vitro human embryos till day 13 post-fertilization
• What kind of new interventions this may allow on our body (including human embryos and human brain) or on our environment?
• The debate is not new but genome editing is now precise, efficient, rapid and not expensive
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“ I would first like to stress that I would be failing in my duty as a scientist if I did not
suggest what might become possible with the new genome editing techniques.
These include some applications involving heritable germline alterations that might be
achievable in the near future; others will be along way off or will never be possible.
However, just because I am voicing these suggestions does not mean that I advocate
that they should be done.
Not only does our scientific knowledge fall short in many cases, but the decision as whether to
go ahead with any specific application is not one for scientists to make alone.”
Robin Lovell-Badge Paris 27-04-2016
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Genome editing redefined
Adapted from R. Barrangou & J. Doudna Nature Biotechnology 34, 933–941 (2016)
Deletions using HDR with a template)
Insertions
Knockouts (using NHEJ-mediated DSB repair)
Transcriptional activation (using dCas9 tethered to a transcriptional activator, i.e. VP64)
Transcriptional repression (dCas9, fused to a repressor i.e KRAB)
Fusion protein delivery
Imaging (using fluorophores)
Epigenetic state alteration
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Mechanism of action of CRISPR–Cas immune systems.
Barangou & Doudna 2016
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1. Basic understanding of human biology: role of specific genes or LncRNA. 2. To create models of human disease in vitro: genetic disorder, cancer.. 3. To treat disease targeting somatic cells. 4. Germline changes to avoid/prevent genetic disease. 5. Germline alterations to give “genetic enhancement”.
Some potential applications for genome editing of human cells
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FOSTERING RESPONSIBLE RESEARCH WITH
CRISPR-Cas9
European Experts Meeting
March 16th, 2016
Biopark Paris
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Purposes of the 16th march 2016
• Rising a european concertation
• Exchange views and present state of regulatory discussions
• Define a process to raise shared guidelines at the european level
• Set-up an advisory group of the european research institutions to
survey and report on the rapid progress and emerging ethical, legal
and societal impacts of genome editing technologies
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What means “Genome editing”?
• Concept associated with the word « editing » : correction, enhancement, improvement but not modification, alteration (in French Modification ciblée du génome = targeted modification of the genome)
• Not new but easier, faster, cheaper and highly efficient
• High speed growing field, race in science (including credits for honors) and an economical challenge with IP fights and big investments: may we control this rapid and multiform dissemination?
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Ethical questions
• Human applications versus animal versus plants
• Research versus medical and/or commercial use
• Transmission/dissemination
• Precautionary principle
• I/ Application of this technology to humans, which essentially raises the question of changes in the germ line;
• II/ Application of this technology to animals, especially to “harmful” species, which raises the question of a possible lateral gene transfer and the emergence of irreversible damage to biodiversity; additional: ethic of animal experimentation (muscle dogs, mini-pigs..)
• III/ Risk of damage to the environment.
• IV/ Genetic Engineering: what is a GMO vs Micro Organism Genetically Engineered (MOGE)
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Common Technical risks
• On-target: create a mutation instead of a disruption
• Off-target: mutation in an untargeted place
• cleavage of a single DNA strand, which could become
uncontrolled and may induce mosaisism or chimerism in the
progeny of the modified cell.
• the targeted mutation is counterbalanced by other
adaptations of genome expression, and that its correction
results in a new balance with unknown effects.
• long-term consequences of the correction must be assessed.
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Ethical Issues Relative to Transmissible DNA
Direct Action on the Inheritance of Certain
Genetic Traits using Gene drives
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Some ethical questions about gene drive
1- the risk of transversal diffusion of the guided gene which would contaminate
organism populations other than that of the target population.
2- the environmental impact resulting from the eradication of a “harmful” population/pathogenic to humans, but that may nonetheless play an important role for biodiversity: alteration of the food chain, loss of pollenization, replacement by another pest….
3- the risk of unwanted side effects of the guided gene for man (appearance of other pathologies associated with guided gene).
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Wrap-Up / Proposals
• A moratorium seems implausible because the idea does not seem to be benefitting from the same international consensus as the 1975 Asilomar conference did. • Example Bärbel Friedrich quoting the Leopoldina 2015 report « research should not not be halted or
hampered »
• At the level of our research institutions there are no bio-security issues here that have not already been considered by previous technologies.
• But, it is not because the technic is easy that it should not go through a rigorous benefit/risk evaluation • Katherine Littler (Wellcome Trust) « nothing should be automatically ruled in or out before it had been fully
explored »
• Call for a responsible use of the technic. We need to take care of public expectation and of the present stage of knowledge : a lot remains to be proven. • We also have heard Anne-Sophie Lapointe (patients association) : “we are here for help and hope.”
• DIY movement might need another consideration and scientists should engage discussions with these groups to increase their understanding of potential harms associated with DNA manipulation.
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Foster research to assess the feasibility, the efficacy and the safety of CRISPR technology and similar developments in experimental models
setting-up of a European Steering Committee (ESC) gathering together experts in
different disciplines as diverse as molecular biology, ecology and a variety of social
sciences, to determine the methods, standards and references.
These methods and standards to evaluate:
?acceptable levels and types of off-target effects,
?acceptable levels of mosaicism,
?acceptable levels of epigenetic effects.
The Steering Committee should rely on stakeholders for an open and transparent
process. These stakeholders will include patient organizations, and representatives of
ethics committees and of the economic sector.
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The potential adverse effects of a gene drive ought to be thoroughly evaluated and mitigated before any clinical or environmental trials are undertaken outside the laboratory
Assessment of a benefit-to-harm balance in the long-term, the European Steering Committee will produce * risk analysis matrices, * devise realistic scenarios * will produce recommendations for reversibility strategies in the case of adverse effects harmful for humans or for biodiversity.
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Adapt the ban on all modifications of the germ line nuclear genome for clinical application in human reproduction.
Oviédo convention 1997, art 13: « an intervention seeking to modify the human genome may only be undertaken for preventive, diagnostic or therapeutic purposes and only if its aims is not to introduce any modification in the genome of any descendants »
An institutional objective for the European research institutions involved could be to participate in defining guidelines. This would mean international cooperation with political decision-makers to determine common strategies and define clear positions from the ethical standpoint on what kinds of translational research are admissible and what kinds are not.
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Need to be pro-active to prevent the
technology being hijacked by those with extreme views
Avoid abusing public expectation with overinflated promises
European research institutions should contribute to national and international initiatives addressing questions of freedom of research and of medical ethics. Participation in such international initiatives by experts from developing countries should be greatly promoted and facilitated, since all people worldwide are concerned, and since local experts need to appropriately control unethical research.
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• Raise awareness about the distinction between the care/treatment of human diseases and human enhancement.
• Certain therapeutic promises might engender dystopic expectations.
• The ethical debate surrounding the life sciences partakes in the necessary acculturation of the society to our disciplines and puts science back into the centre of culture and society as a whole.
• creating a monitoring group led by all stakeholders concerned with genome editing mentioned above (i.e. the European Steering Committee, European research ethics committees, patient organizations, European research institutions and political decision-makers) to promote an open debate on the societal aspects of genomic technologies, before a necessary translation into international legislation becomes possible.
The philosophical question that opposes the dynamic and plasticity of any living organism to an inadequate idea of human nature founded solely on biological determinism.
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• Paris 16/03/16 was the Initial step
• White paper to come: « Fostering Responsible Research with Genome
Editing Technologies: a European Perspective »
• Vienna 07/09/16 “Fighting Malaria with CRISPR/Cas9: Ethical implications”
• Buenos-Aires 01/11/16 as a satellite symposium of the GFBR
• India end of february 2017 with ICMR and DBT
• Thanks to François Hirsch, Solveig Fenet, Christiane Druml, Katherine Littler, Fabiana Arzuaga, Adrienne Hunt, Florencia Luna
Thank you for your attention
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But why not use Preimplantation Genetic Diagnosis (PGD) ?
There are situations where this is not possible, or where it is likely to be inefficient:
•Rare individuals homozygous for dominant mutations, such as Huntington’s Disease.
•Rare occasions where both parents are homozygous for a recessive mutation leading to a genetic disease.
•Mutations affecting fertility where the numbers of embryos obtained are very low, and patients might have to go through many rounds of treatment to find a disease free embryo.
•Where more than one harmful mutation or variant allele makes the probability of finding a “disease-free” embryo very low.
The genome editing methods may turn out to be more efficient and perhaps more reliable than PGD.
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Comparison of blastocyst and early post-implantation development between mouse and human.
Adapted from Rossant (2015),
Development 142:9-12
13
Embryonic day
0
0 1 3.5
2-3 5-6
6.5
MOUSE
HUMAN ZGA
ZGA
Embryonic day
RNA expression studies show many differences in gene activity between mouse and human embryos
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Growth factor conditions and stem cells from early mouse and human embryos
Adapted from: Rossant (2015)
Development, 142:9-12
FGF inhibition
GSK3 inhibition
PKC inhibition
TSCs = trophoblast stem cells
XEN cells =
extraembryonic endoderm stem cells
ES cells
Naïve
or
ground
state
ES cells
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• At fertilisation: Coincident with intracytoplasmic sperm injection (ICSI)
• In zygotes: Injection into the cytoplasm of 1-cell fertilised eggs.
• 2-cell to blastocyst stage embryos: Likely to give mosaics, unless efficient delivery methods such as viral vectors are used.
• Postimplantation stages: In theory, a viral vector could be used to infect germ cells in the embryonic gonads.
• Postnatally: (a) maturing eggs in the ovary. Probably inefficient.
(b) spermatogonial stem cells: Can be cultured, manipulated, and clonal analysis carried out in vitro prior to re-introduction into testes (but complications). Can also be genetically manipulated in vivo.
• Via induced-pluripotent stem cells and in vitro-derived gametes
Stages at which genome editing could be used to modify the human germline
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http://nationalacademies.org/gene-editing/index.htm
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Clinical Use: Somatic
Because proposed clinical uses are intended to affect only the individual who receives them, they can be appropriately and rigorously evaluated within existing and evolving regulatory frameworks for gene therapy, and regulators can weigh risks and potential benefits in approving clinical trials and therapies.
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Clinical Use: Germline It would be irresponsible to proceed with any clinical use of germline editing unless and until (i) the relevant safety and efficacy issues have been resolved, based on appropriate understanding and balancing of risks, potential benefits, and alternatives, and (ii) there is broad societal consensus about the appropriateness of the proposed application.
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Rate of off-target versus natural mutations Genome editing induced off-target mutations have to be considered in the context of naturally occurring mutations: Germline:
Average about 60 point mutations per generation – although age of father is a significant factor with twice as many from old (~80) compared to young men (~40).
[Should we ban old men from having children ?]
In addition there are Indels (~3) and CNVs (??)
[ N.B. Somatic cells have a 4 to 25 times higher rate than seen in the germline.] How to check for off-target events if working with early embryos: Single cell sequencing ? ES cells derived from blastomere biopsy? Post-impalntation biopsy ?
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Some ethical issues :
1. The risks of inaccurate (on-target, or off-target) and inefficient (mosaic) editing.
2. Insufficient knowledge of human genes, genomes and genetic variation, and their interaction with environmental factors: The difficulty of predicting effects (positive or negative) that genome editing may have.
3. The obligation to consider implications for the individual born and for future generations who will carry the genetic alteration.
4. Once introduced into the human gene pool, genetic alterations would be difficult to remove and would not remain within any single community or country.
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Some ethical issues :
1. The risks of inaccurate (on-target, or off-target) and inefficient (mosaic) editing.
2. Insufficient knowledge of human genes, genomes and genetic variation, and their interaction with environmental factors: The difficulty of predicting effects (positive or negative) that genome editing may have.
3. The obligation to consider implications for the individual born and for future generations who will carry the genetic alteration.
4. Once introduced into the human gene pool, genetic alterations would be difficult to remove and would not remain within any single community or country.
Need comparisons with natural mutation rates and the risks associated with human reproduction in the absence of genome editing.
If the change being made is to an allele that already exists in human populations, then is this a legitimate concern ?
The alteration might correspond to a novel linkage with flanking gene sequences. Even then, genome editing could be used to reverse the alteration.
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Some ethical issues, continued :
5. The possibility that permanent genetic “enhancements” to subsets of the population could exacerbate social inequities or be used coercively (including by governments).
What constitutes an “enhancement” ? (Generally or in a specific environment.) And would these be different from other types of enhancement already in use ? 6. The moral and ethical considerations in purposefully altering human evolution using this technology.
But could there be situations where this becomes necessary ? 7. The danger of “rogue” clinics offering unsafe, untested, genome editing methods to ‘treat’ or avoid genetic disease or for enhancements, and either praying on desperate individuals or on the ego and foolishness of others – whether for somatic or heritable germline genetic alterations. The real motive being profit.
Robust regulation and oversight are needed.
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de l’Inserm ● Should genome editing be allowed in basic research involving human germline cells (sperm and egg progenitors) and embryos? ● Should only embryos left over from IVF be used in genome-editing research or may embryos be specifically created for research? ● What safety and efficacy thresholds need to be met before the use of genome editing in human applications, somatic and germline, could be considered? ● If such thresholds are met, what uses for genome editing in human reproductive applications might be permissible? ● How can we avoid the problems associated with “rogue” clinics offering unsafe, untested, genome editing methods to ‘treat’ or avoid genetic disease or for enhancements - a problem for both somatic and germline genome editing ?
Some key questions
